Diversity and expression pattern of novel macin genes in the earthworm, Eiseniaandrei

Antimicrobial peptides (AMPs) are key effectors of innate immunity, providing rapid defense against microbial invasion. Among these, macins form a cysteine-stabilized αβ (CSαβ) peptide family originally described in leeches and cnidarians, but their molecular diversity and immune functions remain poorly characterized in earthworms. Here, we report on the molecular structure, spatial expression, and inducibility of three novel macin genes (Ean-macin1/2/3) identified from the earthworm Eisenia andrei. The deduced amino acids possess a signal peptide and a macin domain. The Ean-macins contain eight conserved cysteine residues predicted to form four disulfide bonds, consistent with the macin/defensin-like fold, and display sequence motifs more similar to neuromacins than hydramacins. In situ hybridization revealed that Ean-macin transcripts were predominantly localized in the circular muscle layer, with weaker signals detected in coelomocytes, peritoneal cells, and bundle sheath of the longitudinal muscles. Microbial challenge assays showed distinct inducibility. Ean-macin1/2 transcripts were broadly upregulated at 12 h post-challenge in response to gram-positive and gram-negative bacteria, lipopolysaccharide (LPS), yeast, and zymosan, whereas Ean-macin3 was rapidly induced at 3 h but only by gram-negative, LPS, and yeast. These results suggest functional divergence among Ean-macins, with Ean-macin1/2 acting as broad-spectrum effectors and Ean-macin3 functioning in early immune response.